Nanotechnology

An injectable refrigerated hydrogel for inducing local hypothermia and neuroprotection against traumatic brain injury in mice | Journal of Nanobiotechnology


Preparation of the Pol hydrogel and Pol/T hydrogel

0.25 g of Pol 407 (25% w/v) and 0.5 g of Pol 188 (5% w/v) were added to 1 mL of high-purity water and placed in a refrigerator at 4 °C until they were fully dissolved. The sol-to-pol transition at physiological temperature was measured at 37 °C in a water bath. For the preparation of the Pol hydrogel, 0.25 g of Pol 407 (25% w/v) and 0.5 g of Pol 188 (5% w/v) containing 50 μg of T1AM were mixed in 1 mL of high-purity water and stored at 4 °C until use.

Rheological monitoring of the Pol/T hydrogel

Dynamic rheometry measurements were carried out on a dynamic Discovery HR-2 rheometer (TA Instruments, USA) equipped with electrically heated plates. Hydrogel samples (400 µL) were added to a parallel plate (diameter 40 mm) using a syringe, and the gap betweent the plate was set to 500 μm. The storage modulus (Gʹ) and loss modulus (G″) data were recorded at 4–45 °C using a strain control of 5%.

Micromorphology of the Pol/T hydrogel

The Pol/T hydrogels were frozen to a solid state in liquid nitrogen, and then the samples were critically dried via vacuum freeze dryer for 48 h. The freeze-dried samples were fixed on aluminium plates. After the powder was sputter-coated with gold, the surface morphology of the Pol/T hydrogel was observed by SEM (FEI Teneo VS).

Degradation studies in vivo

For the assessment of Pol hydrogel degradation in vivo, Pol hydrogels (50 µL) loaded with DiR were injected into weight-drop injury (WDI) model mice, and those mice were imaged with a Xenogen IVIS Spectrum optical device (Caliper, USA) to measure the fluorescence intensity of 1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindotricarbocyanine iodide (DiR) at 3, 6, and 12 h. The data are presented as the means ± SDs (n = 3 mice).

Patients and CSF samples

The human study was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (XYFY2020-KL208-01). All patients provided written informed consent, and all specimens were handled and anonymized according to ethical and legal standards. CSF samples were collected from TBI patients at the Affiliated Hospital of Xuzhou Medical University by lumbar puncture.

In vitro and in vivo drug-release from Pol/T hydrogel

To measure the release of T1AM from the Pol/T hydrogels, 500 µL of each Pol/T hydrogel was transferred to a 4 mL centrifuge tube and suspended in 3 mL of cerebrospinal fluid (CSF). The samples were incubated at 37 °C, and the T1AM released from the Pol/T hydrogels was quantified at different time intervals by high-performance liquid chromatography (Waters e2695) and mass spectrometry (Waters UPLC-ESI-TQD). The column was an advanced Hypersil C18 column (250 × 4.6 mm), and the 1 mL/min mobile phase consisted of methanol: water (45:55) with 0.01% trifluoroacetic acid. The mass spectrometer parameters were as follows: capillary, 0.6 kV; cone, 20 V; collision, 20 V; ion source temperature, 150 °C; gas flow, 800 L/h; and cone gas flow, 20 L/h56. The transitions used for T1AM identification and quantification were 355.904–194.5861 and 355.904–212.0184, respectively. The data are presented as the means ± SDs (n = 3 mice/time).

To measure the T1AM content in the tissues, the cerebral hemispheres and hypothalamus on the traumatized side of the mice treated with phosphate buffer saline (PBS) and Pol/T were dissected at 3 h, 6 h and 12 h. The cerebral hemisphere and hypothalamus tissue samples were homogenized and sonicated for 60 min at 4 °C under dark conditions. Then, 800 μL of methanol was added to the tissue samples to precipitate the proteins. The above mixed tissue samples were vortex mixed for 10 min and centrifuged at 13,000 rpm at 4 °C for 5 min. Then, 100 μL of the supernatant was taken for measurement. The detection method was as described above.

TBI animal models

All experimental procedures were approved by the Xuzhou Medical University of China Animal Care and Use Committee. Five-week-old ICR male and female mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The experimental animals were kept isolated from each other in a cage with an independent air supply. The animals were housed 5 per cage before TBI and then housed singly afterward. After receipt from the vendor, the mice were allowed to acclimate for 1 week. The room temperature was controlled at 23 ± 2 °C. Regular light was maintained (12 h day, 12 h night), and a sufficient supply of water and food was maintained. All mice were healthy at the start of the TBI procedure. A small animal anesthesia machine was used, and isoflurane was used for anesthesia. Anesthesia in mice deepens into the surgical stage: respiratory rate decreases, abdominal breathing becomes predominant, muscles fully relax, eyelid reflex disappears, corneal reflex weakens, and there’s no response to toe or finger pinch. The steps for establishing the WDI models were as follows. A 5 mm craniotomy (3 mm posterior and 3 mm lateral from bregma) was performed on the right parietal region. A weight-drop-hitting device (ZH-ZYQ, Anhui Zhenghua Biological Instrument Equipment Co., Ltd., Huaibei, China) with a 4.0 mm diameter footplate was used to induce injury. Forces of impact were produced by a 40 g weight drop from a height of 7.5 cm. A model of the penetrating brain injury (PBI) was established as follows. At the same position, 9 needles were punctured according to a 3 × 3 grid, with a depth of 3 mm, a width of 1 mm and a height of 1 mm.

The Pol hydrogel group and Pol/T hydrogel group were injected with 50 μL hydrogel, and the T1AM group was injected intraperitoneally with 50 mg/kg of T1AM after trauma. The TBI control group did not receive any therapy. The mice in the rewarming group were immediately rewarmed after being injected with 50 μL of Pol/T hydrogel to maintain the brain temperature at 36 °C by heating the probe through the hollow coil for 12 h (Additional file 1: Figure S1). To reduce the effects of the anesthetic on the rewarmed group, the rewarmed group was heated in the light to maintain a normal body temperature. A temperature probe was inserted into the rectum to monitor the body temperature, and the shivering response of the mice was observed. These measures ensured that the brain and body temperature of the mice in the rewarmed group remained constant throughout the procedure and offset the hypothermia caused by anesthesia. The delayed group was injected with 50 μL of Pol/T hydrogel at 6 h postinjury. The sham group underwent right parietal craniotomy but did not undergo any injury. The sham + Pol group or sham + Pol/T group underwent right parietal craniotomy and were injected with 50 μL of Pol or Pol/T hydrogel.

Temperature measurement

Five-week-old male and female ICR mice were used. TBI was induced via the WDI or PBI, and the treatments were administered to the mice individually. Mice were carefully monitored 2 h after TBI surgery in the cages for waking. If a mouse died after TBI but before the end of the 2 h observation period (2 h after TBI), the data were not included in the analysis. Twenty male mice were equally randomized among these 5 groups: (i) Sham, (ii) Control, (iii) T1AM, (iv) Pol, and (v) Pol/T after the administration of TBI. For every 4 contiguous TBI administrations, one mouse was selected from each of the 5 groups. The order in which the mice were selected from the 5 groups was the same, but the starting group changed with each set of 5 mice. For example, for the first set of 5 mice, the group order was i, ii, iii, iv, and v; for the next set of 5 mice, the order was ii, iii, iv, i, and v. Two animal technicians administered the TBI and subsequent treatments. Three animal technicians collected the data from the mice during the 12 h observation period. Two of these three patients also underwent TBI and treatment and thus were not blinded to treatment when the data were recorded; the one who had not participated in the TBI treatment was blinded. The statistical analyst was not blinded to the groups. The data from 4 mice/group (20 total) were analyzed.

The body and brain temperatures of the mice were measured with probes (Omega Engineering, China). Rectal temperature was measured to represent body temperature. To measure the brain temperature of the mice, a hollow coil was fixed on the surface of the cerebral cortex during modeling. The coil was placed in the bone window and attached to the cerebral cortex, and the coil was fixed with a silk thread. The mice in the rewarming group were continuously anesthetized, and a custom-made oropharyngeal airway was used to maintain patchy breathing. The brain was continuously rewarmed by a heating probe through a hollow coil, and the body of the mice was heated by light so that the body temperature of the mice remained normal. Except for the rewarming group, each mouse was artificially fixed without anesthesia each hour, and the temperature probe was measured through a hollow coil fixed on the dural membrane. The brain temperature was measured along with the anal temperature, which was used to represent the body temperature of the mice. All the mice were measured at 1 h intervals for 12 consecutive hours. These data were obtained by a temperature acquisition system controlled by an instrument (Omega Engineering, China).

Vital sign measurement

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences are noted. Twenty male mice were equally randomized among these 5 groups: (i) Sham, (ii) Control, (iii) T1AM, (iv) Pol, and (v) Pol/T after the administration of TBI. Vital signs, including blood pressure (mm/Hg), heart rate (beats/min), respiratory rate (beats/min) and oxygen saturation (%), were recorded by a life sign monitor (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., Huaibei, China). All the mice were measured at 1-h intervals for 12 consecutive hours. Four mice were measured in each group. In the process of measurement, except for the rewarming group, which was continuously anesthetized, the mice were artificially fixed without anesthesia, and the electrodes were connected to the mice. Life sign monitors automatically displayed the relevant vital sign values.

Spectrophotometric assay for hemoglobin content

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences are noted. Twenty-eight male mice were equally randomized among these 7 groups: (i) Sham, (ii) Control, (iii) T1AM, (iv) Pol, (v) Pol/T, (vi) Delay, and (vii) Rewarming after the administration of TBI. The hemoglobin content of brains subjected to traumatic injury and subjected to different treatments was quantified using a spectrophotometric assay with some modifications [40]. At 12 h after TBI, the mice were deeply anesthetized with isoflurane. The mice were perfused transcardially with PBS. Then, the brain was removed from the skull. The sides of the traumatic lateral hemisphere of the brain were separated, weighed and placed into 4 mL EP tubes. Brain tissue was homogenized on ice and sonicated for 1 min. Brain tissue homogenates were centrifuged at 12,000 r/min for 30 min at 4 °C. A standard curve of hemoglobin was constructed in a 96-well plate that contained increasing ratios of hemoglobin standard to Drabkin reagent. The supernatant of the brain tissue homogenate was added to Drabkin reagent and incubated for 15 min for spectrophotometric analysis.

Mouse tissue immunohistochemistry (IHC) staining

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences are noted. Mouse brain tissues were fixed in 4% paraformaldehyde in sodium citrate buffer and embedded in paraffin for histological analysis. The data from 3 mice/group were analyzed. Hematoxylin and eosin (H&E) staining were performed on 20 µm sections cut with a paraffin slicing machine.

Apoptotic cells in brain tissues were detected by a Tunel BOSTER (MK014) according to the manufacturer’s protocol. In brief, brain tissue sections were incubated with 20 µg/mL protease K and rinsed with 0.3% Triton X-100. The TdT-mediated dUTP nick-end labeling (TUNEL) reaction mixture was added to the brain tissue sections for 2 h at 37 °C in a humidified atmosphere in the dark. Apoptosis was measured by diaminobenzidine (DAB) staining.

Paraffin-embedded brain tissue sections were deparaffinized and boiled in sodium citrate buffer. Then, the brain sections were incubated with normal goat serum. After that, each section was incubated with different primary antibodies against B-cell lymphoma-2 (Bcl-2) (Abcam, ab32124), BCL2-Associated X (Bax) (Abcam, ab81083), matrix metalloproteinase-9 (MMP9) (Abcam, ab236494), ionized calcium binding adapter molecule 1 (Iba-1) (Sigma, SAB2702364), glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, 12389 s) at 4 °C for 12 h. The first antibody was diluted to different concentrations as follow: Bcl-2 (1:100), Bax (1:500), MMP9 (1:100), GFAP (1:200), Iba-1 (1:500). Then, the sections were incubated with a goat anti-rabbit secondary antibody (ZSBio, Beijing, China) for 40 min. Finally, each section was stained with DAB. After 3 washes in PBS and mounting with glycerin, a Leica microscope was used for observation of tissue sections, and the number of positive cells was counted. During the counting process, we based cell existence on the presence of the cell nucleus. Only when positive protein expression co-localized with the cell nucleus was it counted as a positive cell. Five fields of nonoverlapping areas around the wound were taken from each section, and 3 mice were included in each group.

Measurement of cerebral edema and Evans blue (EB) extravasation study

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences are noted. The data from 5 mice/group were analyzed. Cerebral edema was determined by measuring the brain water content at 12 h post-TBI. Following anesthesia by isoflurane and decapitation, the mice were sacrificed, and the brains were collected immediately. The ipsilateral percussion side was separated and weighed to obtain the wet weight. Then, the brain specimens were dried in a desiccating oven at 80 °C for 72 h and weighed again to determine their dry weight. The brain water content was calculated using the following formula: (wet weight-dry weight)/(wet weight). BBB permeability was measured by evaluating the extravasation of EB at 12 h post trauma. EB dye (2%, 2 mL/kg) was administered intravenously at 9 h after TBI and then allowed to circulate for 3 h to ensure that the EB dye fully circulated through the blood system. After anesthesia, 100 mL of PBS was perfused through the left ventricle of the heart, and the brain was removed. The ipsilateral percussion side was separated and weighed. Then, the tissue was placed in 4 mL of potassium hydroxide (1 M) and homogenized. The 1 mL mixture was blended with 5 mL of 0.2 M phosphoric acid and acetone (5:12) and centrifuged at 3000 rpm for 30 min. The supernatant was transferred, and the absorbance at 620 nm was detected.

Magnetic resonance imaging examination

Experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding were the same or similar as in experiment of temperature measurement. Differences are noted. Data from 5 mice/group were analyzed. On the 12th h after the TBI, T2-weighted sequence and diffusion-weighted imaging (DWI) was evaluated by small animal-specific 7 T Micro-MR (Bruker, Bio Spin MRI Pharma Scan 7.0 T). The mice were mildly anesthetized with isoflurane (3.5% induction, and 1.5% maintenance). Throughout the process, the life sign monitors continuously monitored vital signs. According to the vital signs, the inhalation flow was adjusted in time to avoid the death of mice. Once death occurred, the experiment was excluded. The T2-weighted MRI parameters were: repetition time: 2500 ms, echo time: 36 ms, slice thickness: 1 mm, field of view: 20 × 20 mm, image matrix: 256 × 256. The DWI parameters were: repetition time: 3000 ms, echo time: 20 ms, slice thickness: 1 mm, field of view: 100 × 110 mm. All MRI were evaluated by a proficient neuroimaging doctor who was blinded to the study design. Based on the T2 images, the area of edema was measured using Image-J software. The ADC value size was automatically calculated by the Paravision 6.1 of the MRI scanner based on the DWI and the DWI image was processed by the Paravision 6.1 with pseudo-color.

Enzyme linked immunosorbent assay (ELISA)

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences are noted. The data from 3 mice/group were analyzed. The mice were anesthetized and euthanized, and the brains were then removed at 12 h after injury. Then, the ipsilateral percussion side was separated and weighed. The tissue was fully homogenized in lysis solution and centrifuged at 12,000 rpm for 10 min. The supernatant was assessed by ELISA kits (Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions.

Neurologic function assessment

The experimental animals, sample size per group, randomization, inclusion and exclusion criteria and blinding methods used were the same or similar to those used for the temperature measurements. Differences were noted. The data from 5 mice/group were analyzed. The Morris water maze experiment was used to evaluate the recovery of spatial memory and learning ability in mice. All mice were tested for 6 days from the 21st day to the 26th day after TBI. During the first 5 d, the mice were randomly placed in the water in a certain quadrant, and the mice could swim freely in the pool until they found the platform to rest. On the 6th day, the platform was removed, and the platform quadrant was randomly selected. All the data collected during the test were recorded and analyzed by a video tracking system (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., Huaibei, China). The residual motor dysfunction was evaluated by the modified neurological severity score (mNSS) and the wire hanging test at 21 d after TBI. The mNSS included rotation, cylinder, corner, and beam tests. In the course of the wire hanging test, the mice were placed on metallic wire, and the wire was inverted. The latency to fall was recorded.

Statistical analyses

All the statistical analyses were performed using SPSS version 16.0, and the statistical graphs were generated with GraphPad Prism 8.0. If the quantitative data conformed to a normal distribution, the data are presented as the means ± SDs. If the quantitative data did not conform to a normal distribution, the data are presented as quartiles (Q50 (Q25, Q75)). Qualitative data are expressed as percentages or constituent ratios. If multiple sets of quantitative data conformed to a normal distribution and homogeneity, statistical significance was analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test and repeated measures one-way ANOVA with post hoc Bonferroni analysis. If multiple sets of quantitative data did not conform to a normal distribution or homogeneity, the statistical significance of differences between multiple groups was analyzed using the Kruskal‒Wallis test.